Warm Needle Acupuncture Attenuates Pain-Related Responses and Inflammation in Knee Osteoarthritis: Evidence for Involvement of TAK1/MAPK Signaling from Human Cartilage and Experimental Models.

BACKGROUND: Warm needle acupuncture (WNA) can improve symptoms in knee osteoarthritis (KOA). However, the molecular changes linked to its effects remain unclear. METHODS: This study combined human cartilage proteomics, a rat model of destabilization of the medial meniscus (DMM), and ex vivo primary chondrocyte analysis. The final clinical analysis included 61 patients with advanced KOA (control, n = 31; WNA, n = 30). Cartilage and synovial fluid were collected after a 21-day preoperative intervention. Cartilage samples underwent four-dimensional data-independent acquisition (4D-DIA) proteomics, followed by hypothesis-driven analyses focusing on mitogen-activated protein kinase (MAPK)-related processes. Differentially expressed proteins (DEPs) were defined as |log2FC| ≥ 1 and nominal P < 0.05. A DMM-induced KOA rat model involving 60 rats was used for in vivo validation. Mechanical pain hypersensitivity was assessed using the von Frey test, and cartilage pathology, MAPK signaling, and inflammatory cytokines were evaluated. Primary chondrocytes were isolated after in vivo treatment. Ex vivo analyses assessed transforming growth factor-β-activated kinase 1 (TAK1)/MAPK signaling and interleukin-6 (IL-6). Intra-articular interleukin-1β (IL-1β) was used as an additional inflammatory challenge. RESULTS: WNA was associated with lower immunoreactivity for p-TAK1, p-JNK, p-ERK, and p-p38 in human KOA cartilage. Synovial fluid IL-6 (P < 0.01), IL-1β (P < 0.05), and tumor necrosis factor-α (TNF-α) (P < 0.05) were also lower. Proteomics identified 475 DEPs. MAPK-focused analyses showed changes involving JNK-, ERK1/ERK2-, and p38 MAPK-related processes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses also identified inflammatory pathways including MAPK, nuclear factor kappa B (NF-κB), interleukin-17 (IL-17), and TNF signaling. In DMM rats, mechanical paw withdrawal thresholds were reduced after model establishment. WNA progressively increased paw withdrawal thresholds during treatment, with significant improvement compared with the Model group after 14 and 21 days of intervention (both P < 0.001). WNA also reduced cartilage damage and OARSI scores (P < 0.001), accompanied by lower TAK1/MAPK signaling and inflammatory cytokine levels. Chondrocytes from WNA-treated DMM rats also showed lower TAK1/MAPK signaling and IL-6 expression. Intra-articular IL-1β attenuated several WNA-associated molecular changes. CONCLUSION: WNA was associated with lower TAK1/MAPK signaling and reduced IL-1β, TNF-α, and IL-6 in KOA. Human cartilage proteomics also showed WNA-associated changes in MAPK-related processes. Together, these findings support the involvement of TAK1/MAPK-related inflammatory signaling in the response to WNA. Direct functional studies are needed to determine the causal roles of TAK1 and individual MAPK branches. TRIAL REGISTRATION: International Traditional Medicine Clinical Trial Registry (ITMCTR), registration number: ITMCTR2025002585.